This proposal is a response to PA-07-320, Development of Assays for High-throughput Drug Screening (HTS) (R01). The Program Announcement is a component of the Molecular Libraries Initiative, part of the NIH Roadmap for Medical Research, and supports the development of innovative assays that may ultimately be adapted for automated screening of molecular libraries. Our specific aim is to produce and validate screens for identification of drugs from molecular libraries which exert their actions by a newly appreciated therapeutic approach-namely, control of post-translational protein folding and, as a consequence, cellular trafficking and rescue of mutants. G protein coupled receptors (GPCRs) are frequently targeted in library screening, yet this approach generally relies on screens that identify agonists or antagonists and would have missed many of the drugs that will be identified in the proposed screens. Our approach identifies drugs with a significant degree of novelty in therapeutic approach, relying on cellular mechanisms that are not currently represented in the Molecular Libraries assay pipeline; this offers an untapped opportunity for use of the HTS approach. This proposal addresses one of the specific areas that the PA identifies as relevant, assays for molecular chaperones or molecules that improve the post- translational targeting, folding or assembly of proteins, especially involving mutant proteins responsible for inborn errors of metabolism... (or) rare diseases. In the planned studies we will develop and characterize assays for pharmacological chaperones that improve the post-translational folding and targeting of two mutant GPCRs which cause human disease. The products of the proposed HTS assays will be useful for treatment of two rare diseases, nephrogenic diabetes insipidus and hypogonadotropic hypogonadism, each caused by an inborn error. These screens will also serve as prototypes for identification of other therapeutic molecules, especially those involving GPCRs. Many diseases are now understood to be caused by protein misfolding. Development of such assays is important and novel since the extensive use of agonist/antagonist screens alone means that useful chemical structures with the ability to control trafficking (without receptor agonism or antagonism) may already be present in existing libraries, but have not been identified using existing methods. PUBLIC HEALTH RELEVANCE: We will develop and characterize assays for pharmacological chaperones that improve the post- translational folding and targeting of two mutant receptors which cause human diseases, including nephrogenic diabetes insipidus and hypogonadotropic hypogonadism, each caused by an inborn genetic error. These screens will also serve as prototypes for identification of other therapeutic molecules, and are important because existing screens would have missed potentially valuable drugs already present in chemical libraries.